Receptor linked protein tyrosine phosphatases
AssigneeDana-Farber Cancer Insitute
InventorsChristin Frederick, Haruo Saito
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Abstract
The crystal structures of CD45 and LAR, described herein, provide a basis for kinetic and functional studies. Identification of the crystal structures of cellular molecules is important in to determine functional roles in immunity, phosphorylation events, disease initiation mechanism. The isolated crystals and methods for crystallization thereof, are also important in identifying small molecule interactions with cellular molecules for new drug discovery
Claims53
A crystal comprising CD45 or LAR molecular structures wherein the crystal effectively diffracts X-rays for the determination of at least one of the structures to a resolution of about 5 Angstroms to about 2 Angstroms or less.
A method of making crystals comprising:
- cloning of CD45 or LAR molecules from cells producing said molecules into suitable expression vectors;
- and,
- contacting said expression vectors with suitable host cells;
- wherein,
- said host cells express CD45 or LAR gene products;
- and,
- purifying said gene products;
- wherein,
- said purified gene products are mixed with reservoir solutions;
- and,
- growing crystals of said gene products by hanging drop micro vapor diffusion;
- wherein,
- said crystals effectively diffract X-rays for the determination of said crystal structures to a resolution about 5 Angstroms or less and sufficient to determine atomic co-ordinates of said crystals.
A crystal comprising D1 and D2 PTPase domains, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates to a resolution of at least about 3.0 Angstroms;
- and wherein said crystal has a space group of P1 with the unit cell dimensions of a=86 Å, b=60 Å, c=161 Å, α=90°, β=100° and γ=90°.
A crystal comprising the D1 and D2 PTPase domains, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates to a resolution of about 5 Angstroms or greater;
- and wherein said crystal has a space group of P2(1) with the unit cell dimensions of a=86.0 Å, b=59.7 Å, c=160.0 Å and β=99.9°.
A crystal comprising the D1 and D2 PTPase domains, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates to a resolution of about 5 Angstroms or greater;
- and wherein said crystal has a space group of P2(1) with the unit cell dimensions of a=66.92 Å, b=62.73 Å, c=161.59 Å.
A crystal structure wherein LAR is co-crystallized with phosphate analogues in the presence of phosphopeptide substrates.
A crystal structure wherein CD45 is co-crystallized with phosphate analogues in the presence of phosphopeptide substrates.
A crystal structure of CD45 wherein the crystal comprises mutated amino terminal and carboxy terminal ends.
A method of identifying molecules that bind to a CD45 molecule, the method comprising:
- selecting a potential compound through use of the set of atomic coordinates in FIGS. 4A through D, wherein said selecting is performed in conjunction with computer modeling;
- contacting the potential compound with a CD45 molecule or fragments thereof;
- and,
- measuring the binding affinity of CD45 molecule or fragments thereof;
- and
- co-crystallizing CD45 molecule or fragments thereof;
- wherein a potential compound is identified when the crystal effectively diffracts X-rays for the determination of said structures to a resolution of about 5 Angstroms or greater, sufficient to determine atomic co-ordinates of the crystals.
A method for obtaining structural information of molecule, comprising:
- a) generating an X-ray diffraction pattern from a crystallized molecule or molecular complex;
- and,
- b) applying at least a portion of the structure coordinates to the X-ray diffraction pattern to generate a three-dimensional electron density map of the molecule or molecular complex whose structure is unknown.
Dependencies are inferred from the claim text.